U.S. patent number 9,926,522 [Application Number 13/862,966] was granted by the patent office on 2018-03-27 for culture container for adherent cells and method for producing culture container for adherent cells.
This patent grant is currently assigned to Toyo Seikan Kaisha, Ltd.. The grantee listed for this patent is Toyo Seikan Kaisha, Ltd.. Invention is credited to Yoichi Ishizaki, Masahiro Kuninori, Kyohei Ota, Ryo Suenaga, Satoshi Tanaka, Takahiko Totani.
United States Patent |
9,926,522 |
Totani , et al. |
March 27, 2018 |
Culture container for adherent cells and method for producing
culture container for adherent cells
Abstract
A culture bag for culturing adherent cells is provided without
requiring a highly clean production environment and a complex
production step such as a masking step. An adherent cell culture
vessel that is formed of a polyolefin is configured so that part or
the entirety of the inner surface of the culture vessel has a
static water contact angle of 95.degree. or more, and has an
advancing contact angle and a receding contact angle that satisfy
the inequality "advancing contact angle-receding contact
angle>25.degree." when water runs down along the inner
surface.
Inventors: |
Totani; Takahiko (Kanagawa,
JP), Ishizaki; Yoichi (Kanagawa, JP),
Tanaka; Satoshi (Kanagawa, JP), Suenaga; Ryo
(Kanagawa, JP), Ota; Kyohei (Kanagawa, JP),
Kuninori; Masahiro (Kanagawa, JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
Toyo Seikan Kaisha, Ltd. |
Tokyo |
N/A |
JP |
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Assignee: |
Toyo Seikan Kaisha, Ltd.
(Tokyo, JP)
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Family
ID: |
45938060 |
Appl.
No.: |
13/862,966 |
Filed: |
April 15, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130230914 A1 |
Sep 5, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/JP2011/005590 |
Oct 4, 2011 |
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Foreign Application Priority Data
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Oct 13, 2010 [JP] |
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2010-230543 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M
21/00 (20130101); C12M 23/20 (20130101); C12M
23/14 (20130101); C12M 25/14 (20130101) |
Current International
Class: |
C12M
1/00 (20060101); C12M 1/12 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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101652660 |
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Feb 2010 |
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CN |
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1921450 |
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May 2008 |
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EP |
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3-187853 |
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Aug 1991 |
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JP |
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6-098756 |
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Apr 1994 |
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JP |
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2006-204232 |
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Aug 2006 |
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JP |
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2008-048654 |
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Mar 2008 |
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JP |
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2008-174714 |
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Jul 2008 |
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JP |
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2009-027944 |
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Feb 2009 |
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JP |
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2009-027945 |
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Feb 2009 |
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JP |
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2006/107843 |
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Oct 2006 |
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WO |
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Other References
Office Action in corresponding Japanese Application No. 2010-230543
dated Nov. 25, 2014 (6 pages). cited by applicant .
Notification of Transmittal of Translation of the International
Preliminary Report on Patentability issued in corresponding
International Application No. PCT/JP2011/005590 dated May 16, 2013
(1 page). cited by applicant .
International Preliminary Report on Patentability and Written
Opinion issued in corresponding International Application No.
PCT/JP2011/005590 dated May 8, 2013 (5 pages). cited by applicant
.
Lu et al., "A Comparative Study of the Wettability of Steel,
Carbon, and Polyethylene Fibers by Water," Cement and Concrete
Research, vol. 28, No. 6, pp. 783-786, Apr. 10, 1998 (4 pages).
cited by applicant .
Wang et al.; "Three-dimensional Shape of C6 Glioma Cells on the
Fractal Surface"; Chinese Journal of Biomedical Engineering, vol.
28, No. 2, pp. 280-284; Apr. 2009 (5 pages). cited by applicant
.
International Search Report issued in corresponding International
Application No. PCT/JP2011/005590 dated Dec. 13, 2011, and English
translation thereof (2 pages). cited by applicant .
Office Action in corresponding Chinese application No.
201180049002.4 dated Jan. 13, 2014 (8 pages). cited by applicant
.
Office Action issued in Korean Patent Application No.
10-2013-7007966, dated Apr. 28, 2014 (14 pages). cited by applicant
.
Extended European Search Report issued in corresponding European
Application No. 11832264.3 dated Oct. 18, 2016 (10 pages). cited by
applicant .
"Cellular Attachment to Ultraviolet Ozone Modified Polystyrene
Surfaces," Teare D.O. et al., American Chemical Society Langmuir.,
vol. 16, pp. 2818-2824, Jan. 21, 2000 (7 pages). cited by applicant
.
"Line Energy and the Relation Between Advancing, Receding, and
Young Contact Angles", Tadmor R., American Chemical Society
Langmuir., vol. 20, pp. 7659-7664, Jul. 30, 2004(6 pages). cited by
applicant .
"Guide to Use Plastic Additives," Kogyo Chosakai Publishing Co.,
1st edition, 2nd impression, pp. 61-67, Feb. 15, 1998 (8 pages).
cited by applicant.
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Primary Examiner: Prakash; Gautam
Attorney, Agent or Firm: Osha Liang LLP
Claims
The invention claimed is:
1. An adherent cell culture vessel comprising: an inner layer,
wherein the inner layer is a layer of a polyolefin that forms an
inner surface of the culture vessel, wherein the polyolefin is
selected from the group consisting of Easy Processing Polyethylene
(EPPE), Linear Low-Density Polyethylene (LLDPE) and polypropylene
(PP), wherein the polyolefin comprises a phenol-based antioxidant
comprising the following formula: ##STR00001## wherein the
polyolefin is irradiated with UV rays including a wavelength of 254
nm at a cumulative dose of at least 5 J/cm.sup.2, and wherein a
part or entirety of the inner surface of the culture vessel has a
static water contact angle of 95.degree. or more, and has an
advancing contact angle and a receding contact angle that satisfy
an expression (1) when water runs down along the inner surface of
the culture vessel, Advancing contact angle-receding contact
angle>25.degree. (1).
Description
TECHNICAL FIELD
The invention relates to a cell culture technique. In particular,
the invention relates to an adherent cell culture vessel for
culturing adherent cells that require a vessel wall or the like as
a scaffold during culture, and a method for producing an adherent
cell culture vessel.
BACKGROUND ART
In recent years, it has been desired to efficiently culture a large
amount of cells, tissue, microorganisms, or the like in an
artificial environment in the fields of drug production, gene
therapy, regenerative medicine, immunotherapy, and the like.
A culture vessel (culture bag) may be charged with cells and a
culture medium, and the cells may be automatically cultured in
large quantities in the closed system.
Such a culture vessel is required to exhibit gas permeability and
durability necessary for cell culture, heat-seal strength necessary
for forming a bag, and the like, and has been formed using a
polyolefin resin that exhibits these properties.
When using a culture vessel that is formed of a polyolefin resin,
floating cells can be easily cultured, but it has been difficult to
efficiently culture adherent cells that require a vessel wall or
the like as a scaffold during culture. Specifically, it is
necessary for the culture surface to have moderate wettability and
hydrophilicity in order to allow adherent cells to adhere to the
wall of the culture vessel. However, since the surface of a culture
vessel formed of a polyolefin resin is hydrophobic, adherent cells
cannot sufficiently adhere to the culture surface, and it has been
difficult to achieve high proliferation efficiency.
In order to solve the above problem, a technique that subjects the
culture surface of a film that forms a culture vessel to a
hydrophilization treatment using a corona discharge method (see
Patent Document 1) or a UV-ozone method (see Patent Documents 2 and
3) before forming a culture vessel in the shape of a bag has been
proposed.
Patent Document 1: JP-A-6-98756
Patent Document 2: JP-A-2009-27944
Patent Document 3: JP-A-2009-27945
SUMMARY OF THE INVENTION
Technical Problem
However, since the heat-seal strength of the film significantly
decreases as a result of performing the hydrophilization treatment,
it may be difficult to appropriately form a culture vessel in the
shape of a bag.
In order to solve the above problem, Patent Document 3 proposes
performing the UV-ozone treatment while masking the heat seal area
of the film. However, this method requires a complex production
step.
Since the culture surface is exposed during the hydrophilization
treatment, it has been difficult to produce a culture vessel in a
sterile manner. Therefore, a highly clean production environment
may be required.
The inventors of the invention conducted extensive studies, and
found that the adhesion rate of adherent cells to the culture
surface in a state in which a culture medium is contained in the
culture vessel can be significantly improved without hydrophilizing
the culture surface by applying specific UV rays to a resin that
forms the culture vessel. This finding has led to the completion of
the invention.
An object of the invention is to provide an adherent cell culture
vessel that ensures that the cell adhesion rate can be improved,
and adherent cells can be easily cultured by increasing the
hysteresis (advancing contact angle-receding contact angle when
water runs down) to be larger than a given value while ensuring
that part or the entirety of the inner surface of the culture
vessel formed of a polyolefin has a high static water contact angle
(i.e., exhibits hydrophobicity), and a method for producing an
adherent cell culture vessel.
Solution to Problem
An adherent cell culture vessel according to one aspect of the
invention includes a single-layer or multi-layer film or sheet that
includes a layer of a polyolefin as at least an innermost layer,
part or entirety of an inner surface of the culture vessel having a
static water contact angle of 95.degree. or more, and having an
advancing contact angle and a receding contact angle that satisfy
the following expression (1) when water runs down along the inner
surface of the culture vessel.
A method for producing an adherent cell culture vessel according to
another aspect of the invention includes applying UV rays having a
wavelength other than an ozone-generating wavelength to a
single-layer or multi-layer film or sheet that includes a layer of
a polyolefin as at least an innermost layer so that part or
entirety of an inner surface of a culture vessel formed of the film
or sheet has a static water contact angle of 95.degree. or more,
and has an advancing contact angle and a receding contact angle
that satisfy the following expression (1) when water runs down
along the inner surface of the culture vessel.
A method for producing an adherent cell culture vessel according to
another aspect of the invention includes applying UV rays having a
wavelength other than an ozone-generating wavelength to pellets of
a polyolefin, forming a single-layer or multi-layer film or sheet
that includes a layer of the polyolefin as at least an innermost
layer, and
forming an adherent cell culture vessel using the film or sheet so
that part or entirety of an inner surface of the culture vessel has
a static water contact angle of 95.degree. or more, and has an
advancing contact angle and a receding contact angle that satisfy
the following expression (1) when water runs down along the inner
surface of the culture vessel. Advancing contact angle-receding
contact angle>25.degree. (1)
Advantageous Effects of the Invention
These aspects of the invention make it possible to produce a
culture vessel that ensures that adherent cells can be efficiently
cultured, without requiring a highly clean production environment
and a complex production step such as a masking step.
BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1A and 1B are views showing an improvement in cell adhesion
rate and suppression of a decrease in heat-seal strength achieved
by an adherent cell culture vessel according to the invention.
FIG. 2 is a view showing a change in properties (contact angle) of
a film that forms an adherent cell culture vessel due to a UV
irradiation treatment.
FIG. 3 is a view showing a change in properties (hysteresis) of a
film that forms an adherent cell culture vessel due to a UV
irradiation treatment.
FIG. 4 is a view showing a process for producing an adherent cell
culture vessel according to the invention.
FIGS. 5A, 5B, and 5C are views showing the UV irradiation target
(film, culture vessel, or film during formation) when producing an
adherent cell culture vessel according to the invention.
FIG. 6 is a view showing a cell adhesion mechanism when culturing
cells using an adherent cell culture vessel according to the
invention.
FIG. 7 is a view showing a contact angle measurement method and a
hysteresis measurement method used in the examples and comparative
examples.
FIG. 8 is a view showing the experimental conditions, the cell
adhesion rate, the hysteresis, and the heat-seal strength in the
examples and comparative examples.
FIG. 9 is a view showing the relationship between the hysteresis
and the cell adhesion rate in the examples and comparative
examples.
DESCRIPTION OF EMBODIMENTS
An adherent cell culture vessel and a method for producing an
adherent cell culture vessel according to several embodiments of
the invention are described in detail below.
Culture Vessel
1. Material
An adherent cell culture vessel according to one embodiment of the
invention is produced by forming a film using a flexible packaging
material, and forming a bag using the film. The culture vessel is
charged with a culture medium and cells, and the cells are cultured
in the culture vessel. The culture vessel may be pressed using a
plate, or the cells cultured in the culture vessel may be
micrographed at regular intervals, for example. Therefore, it is
desired that a material for forming the culture vessel exhibit gas
permeability and durability necessary for culturing the cells,
exhibit high heat-seal strength, and also exhibit transparency that
allows the contents of the culture vessel to be observed.
A polyolefin resin (e.g., polyethylene or polypropylene) may
suitably be used as a material that meets such requirements.
The film may be a single-layer film, or may be a multi-layer film
that includes two or more layers. A multi-layer film may be formed
by stacking different polyolefin resins of different type or brand.
The film includes a layer of a polyolefin as at least the innermost
layer, and may include a layer formed of a resin other than a
polyolefin. The film may be formed in the form of a sheet.
It is preferable that the material for forming the adherent cell
culture vessel according to one embodiment of the invention include
a polyolefin resin and an antioxidant. The hysteresis of the film
that forms the adherent cell culture vessel can be increased by
applying specific UV rays to the material that includes an
antioxidant, and the hydrophilicity of the culture surface can be
improved when the culture vessel is charged with a culture medium,
so that adhesion of cells can be improved (described later).
The antioxidant is not particularly limited. It is preferable to
use a phenol-based antioxidant or a phosphorus-based antioxidant
due to low cytotoxicity and a capability to advantageously increase
the hysteresis.
2. UV Irradiation Treatment
The properties of the film that forms the adherent cell culture
vessel according to one embodiment of the invention are changed by
applying specific UV rays to the polyolefin resin to modify the
properties of the polyolefin resin.
The properties of the culture surface of the culture vessel can be
changed by forming the culture vessel in the shape of a bag using
the film formed of the polyolefin resin, and applying UV rays from
the outer side of the culture vessel.
The culture surface does not show a significant change in contact
angle (i.e., the culture surface is not hydrophilized) when UV rays
are applied to the culture vessel (film) (described later).
On the other hand, the hysteresis (contact angle hysteresis) of the
culture surface of the adherent cell culture vessel according to
one embodiment of the invention increases as a result of applying
specific UV rays. As a result, the culture surface exhibits high
hydrophilicity when the culture vessel is charged with a culture
medium, and the cell adhesion rate is improved.
In one embodiment of the invention, a UV source that does not apply
light having an ozone-generating wavelength (185 nm) is used for
the UV irradiation treatment. For example, a low-pressure mercury
lamp that has a dominant wavelength of 254 nm and can cut the
ozone-generating wavelength using a quartz tube or the like may
suitably be used.
The adherent cell culture vessel according to one embodiment of the
invention is thus configured so that the cell adhesion rate can be
improved without causing the culture surface to show a significant
change in contact angle by applying UV rays having a wavelength
other than the ozone-generating wavelength to the polyolefin
resin.
Specifically, the adherent cell culture vessel according to one
embodiment of the invention can be produced without exposing the
culture surface since the culture surface is not hydrophilized.
Moreover, the film shows only a small decrease in heat-seal
strength. Therefore, the culture vessel can be successfully
produced even if UV rays are applied to the film before forming the
culture vessel (bag) using the film.
In contrast, the UV-ozone method disclosed in Patent Document 2
hydrophilizes the culture surface by applying UV rays having the
ozone-generating wavelength (i.e., the contact angle decreased from
104.degree. to 71.degree. in the examples of Patent Document 2). It
is considered that ozone derived from oxygen in air reacts directly
with the surface of the material, and the culture surface is
hydrophilized by applying UV rays having the ozone-generating
wavelength. In Patent Document 2, a resin that is easily
hydrophilized, ensures hydrophilization stability, and has a high
glass transition temperature is used as the vessel material. Since
the molecular chain of such a material does not move at room
temperature, it is difficult for a hydrophilic group to enter the
resin after the surface has been hydrophilized, so that
hydrophilization stability is obtained.
The adherent cell culture vessel according to one embodiment of the
invention is formed using polyethylene or polypropylene. Since the
molecular chain of polyethylene or polypropylene can move at room
temperature, and thermodynamic stability is obtained in air when a
hydrophilic group is present inside the resin rather than the
surface of the resin, a hydrophilic functional group rarely moves
to the surface of the resin when the amount of hydrophilic
functional group is small. Therefore, the surface of the resin is
hydrophilized to only a small extent.
3. Cell Adhesion Rate
The inner surface of the adherent cell culture vessel according to
one embodiment of the invention shows a remarkably improved cell
adhesion rate. FIG. 1A shows the cell adhesion rate of the adherent
cell culture vessel according to one embodiment of the invention
that was subjected to the UV irradiation treatment (Example 1), and
the cell adhesion rate of a culture vessel that was produced in the
same manner as the adherent cell culture vessel according to one
embodiment of the invention, except that the UV irradiation
treatment was not performed (Comparative Example 1). The cell
adhesion rate refers to the ratio (%) of the number of adhering
cells to the total number of floating cells and adhering cells
after the adherent cells have been cultured for 28 hours.
As shown in FIG. 1, the cell adhesion rate increased from 3.2% to
80.8% due to the UV irradiation treatment.
Since the cell adhesion rate can be remarkably improved using the
adherent cell culture vessel according to one embodiment of the
invention, the adherent cell culture efficiency can be
significantly improved.
4. Heat-Seal Strength
The adherent cell culture vessel according to one embodiment of the
invention can suppress a decrease in heat-seal strength that may
occur as a result of improving the cell adhesion rate of the inner
surface of the culture vessel.
FIG. 1B shows the heat-seal strength of the adherent cell culture
vessel according to one embodiment of the invention that was
subjected to the UV irradiation treatment, the heat-seal strength
of a culture vessel that was produced in the same manner as the
adherent cell culture vessel according to one embodiment of the
invention, except that the UV irradiation treatment was not
performed, and the heat-seal strength of a culture vessel that was
produced in the same manner as the adherent cell culture vessel
according to one embodiment of the invention, except that a corona
discharge treatment was performed instead of the UV irradiation
treatment. The heat-seal strength shown in FIG. 1 is based on the
maximum force. FIG. 1B shows the heat-seal strength obtained in
Examples 1, 2, and 5 ("UV treatment"), the heat-seal strength
obtained in Comparative Examples 1, 2, and 7 ("Untreated"), and the
heat-seal strength obtained in Comparative Examples 14, 15, and 16
("Corona treatment").
As shown in FIG. 1, while the culture vessel subjected to the
corona discharge treatment showed a significant decrease in
heat-seal strength as compared with the untreated culture vessel,
the adherent cell culture vessel according to one embodiment of the
invention that was subjected to the UV irradiation treatment showed
only a small decrease in heat-seal strength.
Since a decrease in heat-seal strength can be suppressed using the
adherent cell culture vessel according to one embodiment of the
invention, the adherent cell culture vessel can be appropriately
produced even if the UV irradiation treatment is performed before
forming the culture vessel (bag).
5. Contact Angle
The contact angle of the culture surface is described below with
reference to FIG. 2. The hydrophilicity of the culture surface of a
cell culture vessel has a correlation with the contact angle of the
culture surface. The term "contact angle" used herein refers to an
angle (static water contact angle) formed by a liquid surface and a
solid surface when a stationary liquid comes in contact with a
solid surface (.theta.s in FIG. 2). The solid surface has high
hydrophobicity when the contact angle is large, and has high
hydrophilicity when the contact angle is small.
It is generally considered that the contact angle of the culture
surface suitable for culturing adherent cells is about 60 to
80.degree. (Journal of Biomedical Materials Research, Vol. 28,
783-789 (1994)). When the contact angle is within the above range,
the culture surface has high hydrophilicity, and adherent cells
easily adhere to the culture surface.
FIG. 2 shows the contact angle of the adherent cell culture vessel
according to one embodiment of the invention that was subjected to
the UV irradiation treatment (Example 1 (101.9.+-.1.7.degree.)),
and the contact angle of a culture vessel that was produced in the
same manner as the adherent cell culture vessel according to one
embodiment of the invention, except that the UV irradiation
treatment was not performed (Comparative Example 1
(102.7.+-.1.degree.)).
Specifically, the contact angle of the culture surface changed to
only a small extent (i.e., the culture surface was not
hydrophilized) due to the UV irradiation treatment.
Therefore, the adherent cell culture vessel according to one
embodiment of the invention shows a small decrease in heat-seal
strength in spite of the UV irradiation treatment.
When using a related-art technique, adherent cells cannot
sufficiently adhere to the culture surface when the culture surface
is not hydrophilized. In contrast, the adhesion rate of adherent
cells to the culture surface can be improved by the adherent cell
culture vessel according to one embodiment of the invention without
hydrophilizing the culture surface.
The contact angle of the adherent cell culture vessel according to
one embodiment of the invention is preferably 95.degree. or more.
If the contact angle of the adherent cell culture vessel is less
than 95.degree., the hydrophilicity of the culture surface may
increase, and the heat-seal strength may significantly
decrease.
Since part or the entirety of the inner surface of the adherent
cell culture vessel according to one embodiment of the invention
subjected to the UV irradiation treatment has a contact angle of
95.degree. or more, a decrease in heat-seal strength can be
suppressed.
6. Hysteresis
The hysteresis of the film that forms the cell culture vessel can
be increased by the UV irradiation treatment. The term "hysteresis"
used herein refers to a contact angle hysteresis that indicates the
difference between the advancing contact angle (.theta.a) and the
receding contact angle (.theta.r) (run-down hysteresis
(.theta.a-.theta.r)) when a water droplet runs down along the
culture surface. Specifically, a water droplet is dropped onto a
surface that is supported horizontally, and the surface is
gradually tilted. The hysteresis is calculated based on the
advancing contact angle and the receding contact angle at the time
when the water droplet starts to run down along the surface. The
hysteresis is used as an index that indicates the dynamic
wettability of a surface.
FIG. 3 shows the hysteresis of the adherent cell culture vessel
according to one embodiment of the invention that was subjected to
the UV irradiation treatment (Example 1 (27.8.degree.)), and the
hysteresis of a culture vessel that was produced in the same manner
as the adherent cell culture vessel according to one embodiment of
the invention, except that the UV irradiation treatment was not
performed (Comparative Example 1 (16.1.degree.).
Specifically, the hysteresis of the culture surface significantly
increased (i.e., the dynamic wettability of the culture surface
increased) due to the UV irradiation treatment.
Since the culture surface of the adherent cell culture vessel
according to one embodiment of the invention exhibits high dynamic
wettability due to the UV irradiation treatment although the
culture surface is not hydrophilized, the cell adhesion rate can be
improved.
It is preferable that the advancing contact angle and the receding
contact angle when a water droplet runs down along the culture
surface of the adherent cell culture vessel according to one
embodiment of the invention satisfy the following expression (1).
Advancing contact angle-receding contact angle>25.degree.
(1)
As is clear from the examples, while a sufficient cell adhesion
rate cannot be obtained when the hysteresis is 25.degree. or less,
the cell adhesion rate is significantly improved when the
hysteresis is larger than 25.degree..
The entire adherent cell culture vessel may have a hysteresis of
larger than 25.degree., or only part or the entirety of the inner
surface of the adherent cell culture vessel may have a hysteresis
of larger than 25.degree.. Even when only part of the inner surface
of the adherent cell culture vessel has a hysteresis of larger than
25.degree., the cell adhesion rate can be improved in the part of
the inner surface of the adherent cell culture vessel, so that the
cell proliferation efficiency can be improved.
Method for Producing Adherent Cell Culture Vessel
The adherent cell culture vessel according to one embodiment of the
invention may be produced by a normal method, except that a
polyolefin resin is used as the material, and UV rays having a
wavelength other than the ozone-generating wavelength are applied.
For example, the adherent cell culture vessel according to one
embodiment of the invention may be produced by the following steps
(see FIG. 4).
A film (or sheet) is formed by extrusion molding or hot pressing
using polyolefin resin pellets.
It is preferable to add a phenol-based antioxidant or a
phosphorus-based antioxidant to the polyolefin resin when producing
the resin pellets. When the antioxidant is added to the polyolefin
resin, the hysteresis of the polyolefin resin can be advantageously
increased by performing the UV irradiation treatment.
A bag is then formed using the film to obtain an adherent cell
culture vessel.
The UV irradiation treatment may be performed after forming a bag
using the film to obtain a culture vessel (see FIG. 5A).
Specifically, since the UV irradiation treatment is not performed
to hydrophilize the culture surface, UV rays need not be applied
directly to the culture surface. The hysteresis of the culture
surface can be increased by applying UV rays from the outside of
the closed area formed by the film or sheet. It is also possible to
ensure sterility of the culture surface by applying UV rays from
the outside of the closed area.
The UV irradiation treatment may also be performed on a film or
sheet that is not formed in the shape of a bag (see FIG. 5B). The
UV irradiation treatment may also be performed when winding a film
from an inflation film (see FIG. 5C). The UV irradiation treatment
may also be performed on the resin pellets (see FIG. 4).
Specifically, since UV rays having a wavelength other than the
ozone-generating wavelength are applied to the film, the surface of
the film is not hydrophilized, and a significant decrease in
heat-seal strength of the film does not occur.
Therefore, a bag can be appropriately formed even if the UV
irradiation treatment is performed on the film or sheet before
forming a bag, and the adherent cell culture vessel can be produced
without performing a complex step that masks the heat seal
area.
Adhesion Mechanism
A mechanism by which adherent cells adhere to the adherent cell
culture vessel according to one embodiment of the invention is
described below.
Adherent cells normally adhere to a substrate such as a culture
vessel as described below.
Specifically, adherent proteins (e.g., fibronectin or vitronectin)
contained in a culture medium are adsorbed on the substrate.
Integrin or the like that is present on the surface of the adherent
cells interacts with and adheres to the adherent proteins. The
adherent cells thus adhere to the substrate, and grow.
When the substrate is hydrophobic, the part of the adherent
proteins to which the cells adhere interacts with the surface of
the substrate, and interaction with the cells is inhibited. When
the surface of the substrate is highly hydrophilic, the adherent
proteins are not adsorbed on the substrate, and the adherent cells
cannot adhere to the substrate.
It is conjectured that the hysteresis of the culture surface of the
adherent cell culture vessel increases due to the UV irradiation
treatment without an accompanying change in contact angle, and the
cell adhesion rate is improved for the following reasons.
As illustrated in FIG. 6, ROO. is produced from the polyolefin
resin that forms the adherent cell culture vessel according to one
embodiment of the invention due to the UV irradiation treatment.
ROO. reacts with the antioxidant contained in the polyolefin resin,
so that the ester linkage site of the antioxidant that exhibits a
higher degree of freedom can move due to the environment on the
surface of the material.
Therefore, when the adherent cell culture vessel is charged with a
culture medium, and the culture surface is wetted with water, the
hydrophilic group is exposed on the culture surface, and attracts
water. As a result, the hysteresis increases, and the cell adhesion
rate is improved.
Since the hydrophilic group remains in the resin when the culture
surface of the adherent cell culture vessel according to one
embodiment of the invention comes in contact with air, the culture
surface is hydrophobic, and the contact angle does not change.
According to the above adherent cell culture vessel and the method
for producing the same, the contact angle and the hysteresis of the
culture surface can be set within the specific ranges by applying
UV rays having a wavelength other than the ozone-generating
wavelength to the culture vessel that is formed of the polyolefin
resin, and the cell adhesion rate can be improved without
hydrophilizing the culture surface.
This makes it possible to provide an adherent cell culture vessel
that achieves a high cell adhesion rate without requiring a highly
clean production environment and a complex production step such as
a masking step.
EXAMPLES
In the following examples and comparative examples, the performance
and the properties of the adherent cell culture vessel according to
the embodiments of the invention were evaluated as described below
(see FIGS. 7 to 9).
1. The film-forming method, the UV irradiation method, the corona
treatment method, 2. the cell adhesion performance evaluation
method, 3. the contact angle measurement method, the hysteresis
measurement method, and 4. the heat-seal strength measurement
method employed in the examples and comparative examples are
described below.
1. Film-Forming Method, UV Irradiation Method, and Corona Treatment
Method
A film was extrusion-molded using a Labo Plastomill (manufactured
by Toyo Seiki Seisaku-Sho, Ltd.). In some of the examples and
comparative examples, a film was formed using a hot press
(manufactured by Shoji Tekko Co., Ltd.).
The UV irradiation treatment was performed using a UV lamp
("TUV15W/G15T8" manufactured by Philips, dominant wavelength: 254
nm, the ozone-generating wavelength (185 nm) was cut off using a
quartz tube). The UV irradiation treatment was performed at a
cumulative dose of 5 J/cm.sup.2 when performing the UV irradiation
treatment on the bag or the film. In this case, UV rays were
applied for 2 hours in a state in which the film was positioned
apart from the lamp by 12 cm. The UV irradiation treatment was
performed at a cumulative dose of 10 J/cm.sup.2 when performing the
UV irradiation treatment on the resin pellets. In this case, UV
rays were applied for 2 hours in a state in which the resin pellets
were positioned close to the lamp. The cumulative dose was measured
using a cumulative UV meter "UIT-150" (manufactured by Ushio
Inc.).
The corona treatment was performed using a high-frequency power
supply "CG-102" (manufactured by Kasuga Electric Works Ltd.). The
applied current was set to 3.5 A, the distance between the film and
the electrode was set to 5 mm, and the film moving speed was set to
5 m/min.
2. Cell Adhesion Performance Evaluation Method
Adherent cells to be cultured were provided as described below.
Fetal bovine serum (manufactured by Invitrogen) was added to a
medium "Nutrient mixture F/12 Ham" (manufactured by Sigma-Aldrich)
in an amount of 10%. A CHO-K1 (Chinese hamster ovary) cell line was
cultured on a cell culture dish (diameter: 6 cm, manufactured by
Becton, Dickinson and Company). The resulting adherent cells were
collected from the bottom, and the number of adherent cells was
counted.
The film of each example or comparative example was attached to a
Petri dish (diameter: 6 cm, manufactured by Becton, Dickinson and
Company) in advance. The film subjected to the UV irradiation
treatment after forming the bag was cut, and attached to the Petri
dish.
A mixture (300 .mu.l) of 150 .mu.l of a fresh medium and 150 .mu.l
of the cell suspension collected from the bottom of the cell
culture dish was seeded onto the film in a spot-like manner. The
number of cells seeded onto the film differed depending on each
example and comparative example since the number of cells collected
differed depending on the experiment. The number of cells seeded
onto the film was about 7.2 to 8.9.times.10.sup.4 cells/spot. The
culture area was 2 cm.sup.2.
After 1 day had elapsed (after 24 or 28 hours had elapsed), the
number of floating cells and the number of adhering cells were
counted, and the cell adhesion rate was calculated using the
following expression. Cell adhesion rate=(number of adhering
cells)/(number of floating cells+number of adhering cells) 3.
Contact Angle Measurement Method and Hysteresis Measurement
Method
The contact angle and the hysteresis were measured using a
solid-liquid interface analysis system "DropMaster 700"
(manufactured by Kyowa Interface Science Co., Ltd.).
As illustrated in FIG. 7, 3 .mu.l of purified water was dropped
onto the film, and the contact angle (.theta.s) was measured. The
run-down hysteresis (.theta.a-.theta.r) was then measured as
follows. 30 .mu.l of purified water was dropped onto the film, and
the measurement stage was tilted by 1.degree. every second. The
advancing contact angle (.theta.a) and the receding contact angle
(.theta.r) when the water ran down were calculated by a tangent
method to calculate the run-down hysteresis
(.theta.a-.theta.r).
4. Heat-Seal Strength Measurement Method
The heat-seal strength was measured using a heat seal tester
(manufactured by Tester Sangyo Co, Ltd.). The seal width was set to
10 mm, the pressure was set to 3 kgf/cm.sup.2, and the seal time
was set to 2 seconds. Only the upper seal bar was heated to
140.degree. C.
A tensile test was performed using a precision universal tester
"Autograph AG-1S" (manufactured by Shimadzu Corporation). The width
of a test piece was set to be 15 mm, and the test piece was pulled
in the M.D. direction at a speed of 300 mm/min. The heat-seal
strength was measured by the maximum test strength (N/15 mm).
The examples and the comparative examples are described below with
reference to FIG. 8. In FIG. 8, EPPE stands for easy processing
polyethylene, LLDPE stands for linear low-density polyethylene, PP
stands for polypropylene, and LDPE stands for low-density
polyethylene.
"UV after forming bag" in the column "Treatment" means that UV rays
having a wavelength of 254 nm were applied after forming a bag
using the film, and sealing the four sides of the bag. In this
case, the UV irradiation treatment was performed at a cumulative
dose of 5 J/cm.sup.2.
"Direct UV" in the column "Treatment" means that UV rays having a
wavelength of 254 nm were applied in a state in which the film was
exposed. In this case, the UV irradiation treatment was performed
at a cumulative dose of 5 J/cm.sup.2.
"preUV" in the column "Treatment" means that UV rays having a
wavelength of 254 nm were applied to the resin pellets before
forming the film. In this case, the UV irradiation treatment was
performed at a cumulative dose of 10 J/cm.sup.2.
"Corona treatment" in the column "Treatment" means that the corona
treatment was performed using a high-frequency power supply
"CG-102" (manufactured by Kasuga Electric Works Ltd.) in a state in
which the applied current was set to 3.5 A, the distance between
the film and the electrode was set to 5 mm, and the film moving
speed was set to 5 m/min.
Example 1
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding a resin "Excellen FX CX3007" (EPPE,
manufactured by Sumitomo Chemical Co., Ltd.) that contains an
antioxidant. The outer layer was formed using a resin "Excellen GMH
CB2001" (EPPE, manufactured by Sumitomo Chemical Co., Ltd.) to
obtain a two-layer film.
After forming a bag using the film, the four sides of the bag were
sealed, and UV rays having a wavelength of 254 nm were applied to
the film.
The adherent cells (CHO-K1 (Chinese hamster ovary) cell line)
provided as described above were seeded onto the resulting culture
vessel, and cultured. The initial number of cells was
7.8.times.10.sup.4 cells/spot, and the culture area was 2
cm.sup.2.
After 28 hours had elapsed, the number of floating cells and the
number of adhering cells were counted, and the cell adhesion rate
was calculated. The cell adhesion rate thus calculated was
80.8%.
The contact angle and the hysteresis of the culture surface of the
culture vessel, and the heat-seal strength were measured. The
contact angle was 101.9.degree., the hysteresis was 27.8.degree.,
and the heat-seal strength was 15.8 N/15 mm.
Example 2
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding a resin "Excellen FX CX3502" (EPPE,
manufactured by Sumitomo Chemical Co., Ltd.) that contains an
antioxidant. Experiments were performed in the same manner as in
Example 1, except for the above point. The cell adhesion rate was
87.5%, the contact angle was 103.8.degree., the hysteresis was
35.9.degree., and the heat-seal strength was 18.9 N/15 mm.
Example 3
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding a resin "Excellen GMH CB2001" (EPPE,
manufactured by Sumitomo Chemical Co., Ltd.) that contains an
antioxidant. The outer layer was formed using a resin "Kernel
KS240T" (LLDPE, manufactured by Japan Polyethylene Corporation) to
obtain a two-layer film. Experiments were performed in the same
manner as in Example 1, except for the above point. Note that the
heat-seal strength was not measured. The cell adhesion rate was
86.1%, the contact angle was 100.0.degree., and the hysteresis was
26.5.degree..
Example 4
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding a resin "Kernel KS240T" (LLDPE,
manufactured by Japan Polyethylene Corporation) that contains an
antioxidant. Experiments were performed in the same manner as in
Example 1, except for the above point. Note that the heat-seal
strength was not measured. The cell adhesion rate was 82.0%, the
contact angle was 105.7.degree., and the hysteresis was
35.0.degree..
Example 5
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding a resin "Kernel KS340T" (LLDPE,
manufactured by Japan Polyethylene Corporation) that contains an
antioxidant. Experiments were performed in the same manner as in
Example 1, except for the above point. The cell adhesion rate was
87.1%, the contact angle was 104.3.degree., the hysteresis was
40.9.degree., and the heat-seal strength was 17.3 N/15 mm.
Example 6
A film that forms the single-layer cell culture vessel was formed
by extrusion-molding a resin "Kernel KF283" (LLDPE, manufactured by
Japan Polyethylene Corporation) that contains an antioxidant.
Experiments were performed in the same manner as in Example 1,
except that the initial number of cells was 8.9.times.10.sup.4
cells/spot. Note that the heat-seal strength was not measured. The
cell adhesion rate was 81.0%, the contact angle was 95.6.degree.,
and the hysteresis was 30.0.degree..
Example 7
A film that forms the single-layer cell culture vessel was formed
by hot pressing a resin "Novatec-PP MG3F" (PP, manufactured by
Japan Polypropylene Corporation) that contains an antioxidant.
UV rays having a wavelength of 254 nm were applied in a state in
which the film was exposed.
The adherent cells (CHO-K1 (Chinese hamster ovary) cell line)
provided as described above were seeded onto the resulting culture
vessel, and cultured. The initial number of cells was
7.2.times.10.sup.4 cells/spot, and the culture area was 2
cm.sup.2.
After 24 hours had elapsed, the number of floating cells and the
number of adhering cells were counted, and the cell adhesion rate
was calculated. The cell adhesion rate thus calculated was
82.7%.
The contact angle and the hysteresis of the culture surface of the
culture vessel were measured. The contact angle was 97.3.degree.,
and the hysteresis was 25.9.degree..
Example 8
As the film that forms the single-layer cell culture, "Excellen FX
CX3007" (EPPE, manufactured by Sumitomo Chemical Co., Ltd.) that
contains an antioxidant was used. UV rays having a wavelength of
254 nm were applied to the resin pellets thereof. A film was formed
by hot pressing using the pellets to obtain a culture vessel.
The cells were cultured in the same manner as in Example 7 using
the resulting culture vessel, and the cell adhesion rate was
calculated. The cell adhesion rate thus calculated was 91.9%.
The contact angle and the hysteresis of the culture surface of the
culture vessel were measured. The contact angle was 102.2.degree.,
and the hysteresis was 29.2.degree..
Comparative Example 1
Experiments were performed in the same manner as in Example 1,
except that the resin used in Example 1 was used to form the inner
layer of the cell culture vessel, but the UV irradiation treatment
was not performed. The cell adhesion rate was 3.2%, the contact
angle was 102.7.degree., the hysteresis was 16.1.degree., and the
heat-seal strength was 18.5 N/15 mm.
Comparative Example 2
Experiments were performed in the same manner as in Example 2,
except that the resin used in Example 2 was used to form the inner
layer of the cell culture vessel, but the UV irradiation treatment
was not performed. The cell adhesion rate was 1.9%, the contact
angle was 103.2.degree., the hysteresis was 17.0.degree., and the
heat-seal strength was 20.5 N/15 mm.
Comparative Example 3
Experiments were performed in the same manner as in Example 3,
except that the resin used in Example 3 was used to form the inner
layer of the cell culture vessel, but the UV irradiation treatment
was not performed. The cell adhesion rate was 20.7%, the contact
angle was 100.1.degree., and the hysteresis was 17.8.degree..
Comparative Example 4
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding a resin "Excellen GMH CB5002" (EPPE,
manufactured by Sumitomo Chemical Co., Ltd.) that does not contain
an antioxidant. The outer layer was formed using a resin "Kernel
KM262" (LLDPE, manufactured by Japan Polyethylene Corporation) to
obtain a two-layer film.
Experiments were performed in the same manner as in Example 3,
except that UV irradiation treatment was not performed. The cell
adhesion rate was 18.6%, the contact angle was 101.0.degree., and
the hysteresis was 15.6.degree..
Comparative Example 5
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding the resin used in Comparative Example
4. The outer layer was formed using the resin used in Comparative
Example 4 to obtain a two-layer film.
After forming a bag using the film, the four sides of the bag were
sealed, and UV rays having a wavelength of 254 nm were applied to
the film. Experiments were performed in the same manner as in
Comparative Example 4, except that UV irradiation treatment was
performed as described above. The cell adhesion rate was 12.5%, the
contact angle was 100.4.degree., and the hysteresis was
17.1.degree..
Comparative Example 6
Experiments were performed in the same manner as in Example 4,
except that the resin used in Example 4 was used to form the inner
layer of the cell culture vessel, but the UV irradiation treatment
was not performed. The cell adhesion rate was 13.5%, the contact
angle was 106.0.degree., and the hysteresis was 21.9.degree..
Comparative Example 7
Experiments were performed in the same manner as in Example 5,
except that the resin used in Example 5 was used to form the inner
layer of the cell culture vessel, but the UV irradiation treatment
was not performed. The cell adhesion rate was 17.6%, the contact
angle was 103.6.degree., the hysteresis was 25.0.degree., and the
heat-seal strength was 17.5 N/15 mm.
Comparative Example 8
Experiments were performed in the same manner as in Example 6,
except that the resin used in Example 6 was used to form the
single-layer cell culture vessel, but the UV irradiation treatment
was not performed. The cell adhesion rate was 39.0%, the contact
angle was 96.8.degree., and the hysteresis was 15.0.degree..
Comparative Example 9
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding a resin "Kernel KM262" (LLDPE,
manufactured by Japan Polyethylene Corporation) that does not
contain an antioxidant. The outer layer was formed using a resin
"Excellen GMH CB5002" (EPPE, manufactured by Sumitomo Chemical Co.,
Ltd.) to obtain a two-layer film.
Experiments were performed in the same manner as in Example 3,
except that UV irradiation treatment was not performed. The cell
adhesion rate was 16.0%, the contact angle was 100.9.degree., and
the hysteresis was 15.6.degree..
Comparative Example 10
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding the resin used in Comparative Example
9. The outer layer was formed using the resin used in Comparative
Example 9 to obtain a two-layer film.
After forming a bag using the film, the four sides of the bag were
sealed, and UV rays having a wavelength of 254 nm were applied to
the film. Experiments were performed in the same manner as in
Comparative Example 9, except that UV irradiation treatment was
performed as described above. The cell adhesion rate was 17.3%, the
contact angle was 100.8.degree., and the hysteresis was
16.2.degree..
Comparative Example 11
A film that forms the single-layer cell culture vessel was formed
by extrusion-molding a resin "UBE Polyethylene L719" (LDPE,
manufactured by Ube-Maruzen Polyethylene Co., Ltd.) that does not
contain an antioxidant.
Experiments were performed in the same manner as in Example 3,
except that UV irradiation treatment was not performed. The cell
adhesion rate was 53.0%, the contact angle was 102.4.degree., and
the hysteresis was 15.0.degree..
Comparative Example 12
A film that forms the single-layer cell culture vessel was formed
by extrusion-molding the resin used in Comparative Example 11.
After forming a bag using the film, the four sides of the film were
sealed, and UV rays having a wavelength of 254 nm were applied to
the film. Experiments were performed in the same manner as in
Comparative Example 11, except that UV irradiation treatment was
performed as described above. The cell adhesion rate was 47.9%, the
contact angle was 102.3.degree., and the hysteresis was
17.8.degree..
Comparative Example 13
Experiments were performed in the same manner as in Example 7,
except that the resin used in Example 7 was used to form the
single-layer cell culture vessel, but the UV irradiation treatment
was not performed. The cell adhesion rate was 21.1%, the contact
angle was 99.5.degree., and the hysteresis was 16.1.degree..
Comparative Example 14
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding the resin used in Example 1. The outer
layer was formed using the resin used in Example 1 to obtain a
two-layer film.
The corona discharge treatment was performed in a state in which
the film was exposed, and the contact angle and the heat-seal
strength were measured. The contact angle was 87.6.degree., and the
heat-seal strength was 11.3 N/15 mm.
Comparative Example 15
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding the resin used in Example 2. The outer
layer was formed using the resin used in Example 2 to obtain a
two-layer film.
The corona discharge treatment was performed in a state in which
the film was exposed, and the contact angle and the heat-seal
strength were measured. The contact angle was 91.5.degree., and the
heat-seal strength was 10.6 N/15 mm.
Comparative Example 16
A film that forms the inner layer of the cell culture vessel was
formed by extrusion-molding the resin used in Example 5. The outer
layer was formed using the resin used in Example 5 to obtain a
two-layer film.
The corona discharge treatment was performed in a state in which
the film was exposed, and the contact angle and the heat-seal
strength were measured. The contact angle was 85.8.degree., and the
heat-seal strength was 9.2 N/15 mm.
FIG. 9 shows the relationship between the cell adhesion rate and
the hysteresis in the examples and the comparative examples.
In FIG. 9, the black circles indicate the results for the culture
vessels obtained in the examples in which the resin that contains
an antioxidant was used, and UV rays having a wavelength of 254 nm
were applied. The white circles indicate the results for the
culture vessels obtained in the comparative examples in which the
resin that contains an antioxidant was used, and UV rays were not
applied. The black squares indicate the results for the culture
vessels obtained in the comparative examples in which the resin
that does not contain an antioxidant was used, and UV rays having a
wavelength of 254 nm were applied. The white squares indicate the
results for the culture vessels obtained in the comparative
examples in which the resin that does not contain an antioxidant
was used, and UV rays were not applied.
The contact angle of the inner surface of these culture vessels was
95.degree. or more (i.e., the inner surface was not hydrophilized
(see Examples 1 to 8 and Comparative Examples 1 to 13 in FIG.
8)).
The cell adhesion rate increased rapidly when the hysteresis
exceeded 25.degree..
It was thus confirmed from the above results that it is preferable
to produce an adherent cell culture vessel by utilizing a
polyolefin resin (e.g., polyethylene or polypropylene) that
contains an antioxidant, and applying UV rays having a wavelength
other than the ozone-generating wavelength.
This makes it possible to produce a culture vessel that has a
culture surface having a contact angle of 95.degree. or more and a
hysteresis of larger than 25.degree.. A high cell adhesion rate can
be achieved by culturing adherent cells using the culture
vessel.
The invention is not limited to the above embodiments and examples.
Various modifications may be made without departing from the scope
of the invention.
Although the CHO-K1 cell line was used in the examples as the
adherent cells, the adherent cells are not limited thereto. It is
also possible to use other adherent cells. The type of the culture
medium, the film-forming method, the bag-forming method, and the
like may be appropriately changed. Although a low-pressure mercury
lamp that emits UV rays having a wavelength other than the
ozone-generating wavelength was used in the embodiments and the
examples as the UV source, the UV source is not limited thereto as
long as a culture surface having a contact angle of 95.degree. or
more and a hysteresis of larger than 25.degree. can be formed. For
example, a high-pressure mercury lamp that emits UV rays having a
wavelength of 254 nm may also be used.
INDUSTRIAL APPLICABILITY
The invention may suitably be used when culturing a large amount of
adherent cells using a cell culture vessel.
* * * * *